SNCG antibody and application thereof in tumor treatment

Abstract

The invention provides an SNCG antibody and an application of the SNCG antibody in tumor treatment. The amino acid sequences of the six CDRs of the SNCG antibody comprise: GYTFTDYAIH, GYTFTDYAIH, GYTFTDYAIH, aIDXETGGTAYNQKFKG is used as a template; vAY; kSSQSLLDSDXKTYLN is used as a main body of the device; carrying out LVSKLDS (Low Voltage Stock Stock And WQGTHFPQT is adopted. The SNCG antibody and the bevacizure antibody are combined for application, or the SNCG antibody and the VEGF antibody are recombined to prepare a bifunctional antibody, and the sensitivity of cells to the bevacizure antibody can be improved by blocking the biological function of the SNCG.

Description

Technical Field

[0001] The present invention relates to molecules capable of immunospecifically binding to SNCG, in particular to SNCG antibodies and biologically active fragments derived from the antibodies capable of immunospecifically binding to SNCG, as well as their related applications in tumor treatment, especially in bevacizumab-resistant tumor treatment. Background Art

[0002] γ-Synuclein (SNCG) belongs to the synaptic nuclein family. The family consists of three members: synuclein-α (SNCA), synuclein-β (SNCB) and SNCG. It was first discovered in brain tissue and is a natural small molecule soluble neuroprotein. Due to its involvement in the occurrence and development of neurodegenerative diseases, SNCA and SNCB have been widely studied in a variety of neurological diseases such as Alzheimer's disease, Parkinson's disease, and multiple system atrophy. In recent years, many studies have shown that SNCG is highly expressed in a variety of cancer tissues and cancer patients' body fluids such as serum, saliva, and urine, and is negatively correlated with the patient's prognosis. SNCG promotes the movement, invasion, and metastasis of tumor cells by regulating multiple signaling pathways such as ITGb1-FAK, MKK3 / 6-p38MAPK, PI3K / AKT / ERK, etc. SNCG can also increase the resistance of tumor cells to taxane chemotherapy drugs and radiotherapy by binding to proteins such as ER-α36, HSP90, IGF-IR or activating the ERK1 / 2 signaling pathway. At the same time, SNCG is also an important molecule for maintaining the activity of the TGF-β-Smad-Twist1 axis.

[0003] Therefore, the development of SNCG antibodies has important clinical significance. Summary of the invention

[0004] One object of the present invention is to provide a molecule that specifically binds to SNCG.

[0005] Another object of the present invention is to provide an anti-SNCG antibody.

[0006] Another object of the present invention is to provide related applications of SNCG antibodies.

[0007] In order to develop a binding molecule that specifically binds to SNCG, the present invention successfully isolated a mouse SNCG antibody from an immunized mouse. On this basis, the present invention further provides a humanized antibody.

[0008] According to one aspect of the present invention, the present invention provides a molecule that can immunospecifically bind to SNCG, and is therefore also referred to as an SNCG binding molecule in the present invention. Specifically, the molecule includes an SNCG antibody or a biologically active fragment (antigen binding fragment) derived from the antibody that can immunospecifically bind to SNCG, wherein the amino acid sequence of the six CDRs of the SNCG antibody includes:

[0009] Heavy chain CDR1: GYTFTDYAIH (SEQ ID NO: 1);

[0010] Heavy chain CDR2: AIDXETGGTAYNQKFKG (SEQ ID NO: 2), wherein X is P or A;

[0011] Heavy chain CDR3: VAY;

[0012] Light chain CDR1: KSSQSLLDSDXKTYLN (SEQ ID NO: 3), wherein X is G or A;

[0013] Light chain CDR2: LVSKLDS (SEQ ID NO: 4);

[0014] Light chain CDR3: WQGTHFPQT (SEQ ID NO: 5).

[0015] In the present invention, "immunospecific binding" means that if such binding exhibits the specificity and affinity of an antibody binding to its cognate antigen, then a molecule is said to be able to "immunospecifically bind" to another molecule. If such binding involves the antigen recognition site of the immunoglobulin molecule, then the antibody is said to be able to "immunospecifically bind" to a target region or structure ("epitope") of the antigen (and in particular human SNCG). An antibody that immunospecifically binds to a specific antigen may bind to other antigens with lower affinity if the other antigen has been identified by, for example, immunoassay, The antibody may have some sequence or structural similarity to the antigen recognition site identified by the PCR assay or other assays known in the art, but will not bind to completely unrelated antigens. However, preferably, the antibody (and antigen binding fragment thereof) will not cross-react with other antigens. The antibody may also bind to other molecules in a non-immunospecific manner, such as to Fc receptors (FcRs), by virtue of binding domains in other regions / domains of the molecule that are not involved in the antigen recognition site, such as the Fc region.

[0016] In the present invention, a molecule "capable of immunospecifically binding to SNCG" means that the molecule has the ability to weaken or block the binding of SNCG to the ligand by binding to SNCG.

[0017] According to a specific embodiment of the present invention, the biologically active fragment capable of immunospecifically binding to SNCG derived from the antibody of the present invention comprises the amino acid sequence of the six CDRs of the SNCG antibody.

[0018] As used herein, a sequence "variant" refers to a sequence that differs from the indicated sequence at one or more amino acid residues but retains the biological activity of the resulting molecule (the biological activity is not substantially changed compared to the original sequence).

[0019] "Conservatively modified variants" or "conservative amino acid substitutions" refer to amino acid substitutions known to those skilled in the art, which are made without generally altering the biological activity of the resulting molecule. In general, it is recognized by those skilled in the art that single amino acid substitutions in non-essential regions of a polypeptide do not substantially alter biological activity (see, e.g., Watson et al., Molecular Biology of the Gene, The Benjamin / Cummings Pub. Co., p. 224 (4th ed., 1987)). Such exemplary substitutions are preferably made in accordance with the substitutions shown in Table 1 below:

[0020] Table 1. Exemplary conservative amino acid substitutions

[0021]

[0022]

[0023] The present invention provides antibodies and antigen-binding fragments thereof having 6 CDRs of SNCG antibodies, and also provides variants thereof. The variants may, for example, be derived antibodies and antigen-binding fragments thereof having amino acid residues substituted as listed in the table above. These derived variants include amino acid sequences of variable heavy chains and / or variable light chains that exhibit at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% consistency with the amino acid sequences of the variable heavy chains and / or light chains of the SNCG antibodies. In addition, these derived antibodies and antigen-binding fragments thereof may include CDRs that exhibit at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% consistency with the amino acid sequences of the CDRs of the SNCG antibodies. The determination of the percentage identity of the two amino acid sequences can be determined by BLAST protein comparison.

[0024] In the present invention, the term "substantially" used in the context of the effect of combination or display is intended to indicate that the observed effect is physiological or therapeutically relevant. Thus, for example, if the degree of retardation is physiological or therapeutically relevant (e.g., if this degree is greater than 60% of complete retardation, greater than 70% of complete retardation, greater than 75% of complete retardation, greater than 80% of complete retardation, greater than 85% of complete retardation, greater than 90% of complete retardation, greater than 95% of complete retardation, or greater than 97% of complete retardation), a molecule can substantially block the activity of SNCG. Similarly, if such immunospecificity and characterization are greater than 60% identical, greater than 70% identical, greater than 75% identical, greater than 80% identical, greater than 85% identical, greater than 90% identical, greater than 95% identical, or greater than 97% identical, then a molecule is said to have substantially the same immunospecificity and / or characterization as another molecule.

[0025] According to a specific embodiment of the present invention, the antibody of the present invention may be an animal-derived antibody, or may be a chimeric antibody, a humanized antibody or a human antibody.

[0026] In the present invention, "antibody" is intended to mean an immunoglobulin molecule having a "variable region" antigen recognition site. The term "variable region" is intended to distinguish this domain of the immunoglobulin from domains widely shared by antibodies (e.g., an antibody Fc domain). In the present invention, the term "antigen-binding fragment" of an antibody refers to one or more portions of an antibody that include the complementarity determining region ("CDR") of the antibody and optionally framework residues that include the "variable region" antigen recognition site of the antibody and exhibit the ability to immunospecifically bind to an antigen. Such fragments include Fab', F(ab') and 2 , Fv, single chain (ScFv), and mutants thereof, naturally occurring variants, and fusion proteins comprising the "variable region" antigen recognition site of the antibody and a heterologous protein (e.g., a toxin, an antigen recognition site for a different antigen, an enzyme, a receptor or a receptor ligand, etc.). In the present invention, the term "fragment" refers to a peptide or polypeptide that includes an amino acid sequence of at least 3 or 4 consecutive amino acid residues, at least 10 consecutive amino acid residues, at least 15 consecutive amino acid residues, at least 20 consecutive amino acid residues, at least 25 consecutive amino acid residues, at least 40 consecutive amino acid residues, at least 50 consecutive amino acid residues, at least 60 consecutive amino acid residues, at least 70 consecutive amino acid residues, at least 80 consecutive amino acid residues, at least 90 consecutive amino acid residues, at least 100 consecutive amino acid residues, at least 125 consecutive amino acid residues, at least 150 consecutive amino acid residues, at least 175 consecutive amino acid residues, at least 200 consecutive amino acid residues, or at least 250 consecutive amino acid residues.

[0027] Human chimeric antibodies, humanized antibodies or human antibodies are particularly preferred for use in humans, however, antibodies of animal origin may be advantageously employed for many purposes (e.g., in vitro or in situ detection assays, acute in vivo use, etc.). Fully human antibodies are particularly desirable for therapeutic treatment of human subjects. Human antibodies can be manufactured by a variety of methods known in the art, including the above-mentioned phage display methods using antibody libraries derived from human immunoglobulin sequences.

[0028] In the present invention, a "chimeric antibody" is a molecule in which different parts of the antibody are derived from different immunoglobulin molecules, such as an antibody having a variable region derived from a non-human antibody and a human immunoglobulin constant region. Methods for producing chimeric antibodies are known in the art.

[0029] In the present invention, "humanized antibody" refers to an immunoglobulin comprising a human framework region and one or more CDRs from a non-human immunoglobulin. The non-human immunoglobulin providing the CDR is called a "donor" and the human immunoglobulin providing the framework is called an "acceptor". Constant regions do not need to be present, but if they are present, they must be substantially identical to human immunoglobulin constant regions, i.e., at least about 85%-90%, preferably about 95% or more, identical. Therefore, except for possible CDRs, all parts of humanized immunoglobulins are substantially identical to corresponding parts of natural human immunoglobulin sequences. A humanized antibody is an antibody comprising a humanized light chain and a humanized heavy chain immunoglobulin. For example, a humanized antibody will not contain a typical chimeric antibody because, for example, the entire variable region of a chimeric antibody is non-human. A donor antibody is "humanized" through the process of "humanization" because the resulting humanized antibody is expected to bind to the same antigen against which the donor antibody providing the CDR is directed. For most, humanized antibodies are human immunoglobulins (receptor antibodies), wherein the hypervariable region residues of the receptor are replaced by hypervariable region residues from a non-human species (donor antibody) such as mouse, rat, rabbit or non-human primate with desired specificity, affinity and ability. In some cases, the framework region (FR) residues of human immunoglobulin are replaced by corresponding non-human residues. In addition, humanized antibodies can include residues that are not present in receptor antibodies or donor antibodies. These modifications are made to further improve antibody performance. Usually, the humanized antibody will include substantially at least one and typically all of two variable domains, wherein all or substantially all of these hypervariable regions correspond to those of non-human immunoglobulins, and all or substantially all of FR are those of human immunoglobulin sequences. The humanized antibody optionally will also include at least a portion of an immunoglobulin constant region (Fc), typically that portion of a human immunoglobulin that is immunospecifically bound to an FcγRIIB polypeptide, which has been changed by introducing amino acid residue substitutions, deletions or additions (i.e., mutations).

[0030] In the present invention, the antibody may be monospecific, bispecific, trispecific or a more multispecific antibody.

[0031] According to a specific embodiment of the present invention, the following humanized antibody sequences were designed.

[0032] Heavy Chain:

[0033] Heavy chain 1 (H1) of humanized SNCG antibody:

[0034]

[0035] Heavy chain 2 (H2) of humanized SNCG antibody:

[0036]

[0037] Heavy chain 3 (H3) of humanized SNCG antibody:

[0038]

[0039] Light chain:

[0040] Light chain 1 (L1) of humanized SNCG antibody:

[0041]

[0042] Light chain 2 (L2) of humanized SNCG antibody:

[0043]

[0044] Light chain 3 (L3) of humanized SNCG antibody:

[0045]

[0046] According to a specific embodiment of the present invention, the SNCG binding molecule of the present invention comprises:

[0047] (1) a heavy chain variable region, wherein the heavy chain variable region has an amino acid sequence of any one of SEQ ID NO: 6, SEQ ID NO: 8, and SEQ ID NO: 10, or an amino acid sequence having substantially equivalent functions formed by replacing, deleting, or adding one or more amino acids based on these amino acid sequences; and / or

[0048] (2) a light chain variable region, wherein the light chain variable region has an amino acid sequence of any one of SEQ ID NO:7, SEQ ID NO:9, and SEQ ID NO:11, or an amino acid sequence with substantially equivalent functions formed by replacing, deleting, or adding one or more amino acids on the basis of these amino acid sequences.

[0049] According to a specific embodiment of the present invention, the SNCG binding molecule of the present invention may include any one or more of the humanized antibodies listed in Table 2 below.

[0050] Table 2

[0051]

[0052] In some embodiments of the present invention, the following four humanized antibodies were tested:

[0053] Humanized antibody 1# (h1): the heavy chain variable region has the amino acid sequence shown in SEQ ID NO: 6, and the light chain variable region has the amino acid sequence shown in SEQ ID NO: 7;

[0054] Humanized antibody 2# (h5): the heavy chain variable region has the amino acid sequence shown in SEQ ID NO: 8, and the light chain variable region has the amino acid sequence shown in SEQ ID NO: 9;

[0055] Humanized antibody 3# (h6): the heavy chain variable region has the amino acid sequence shown in SEQ ID NO: 8, and the light chain variable region has the amino acid sequence shown in SEQ ID NO: 11;

[0056] Humanized antibody 4# (h9): the heavy chain variable region has the amino acid sequence shown in SEQ ID NO: 10, and the light chain variable region has the amino acid sequence shown in SEQ ID NO: 11;

[0057] The binding activity of humanized SNCG antibodies h1, h5, h6, and h9 was detected by ELISA, and the results showed that all of them had the activity of specifically binding to SNCG.

[0058] The SNCG antibody of the present invention can improve the efficacy of bevacizumab or overcome bevacizumab resistance. The applicant's research also found that SNCG can also activate VEGF receptors and reduce the sensitivity of cells to bevacizumab. The SNCG antibody of the present invention can be used in combination with bevacizumab, or the SNCG antibody and VEGF antibody can be recombined to prepare a bifunctional antibody; or the sensitivity of cells to bevacizumab can be increased by blocking the biological activity of SNCG.

[0059] When the inventors used immunohistochemistry to detect the expression level of SNCG in colorectal cancer (CRC) tissue, they found that the vascular parts of CRC tissue expressed high levels of SNCG. Further studies have shown that SNCG has an angiogenic effect and can synergize with VEGF. In addition, high SNCG expression is associated with bevacizumab treatment resistance. The inhibitory effect of bevacizumab on cell migration is negatively correlated with the expression and secretion levels of SNCG. The growth inhibitory effect of bevacizumab on SNCG-high-expressing tumor cells is significantly lower than that on low-expressing parental cells, and the tumor inhibition effect has no significant correlation with VEGF levels, but is significantly correlated with the SNCG expression level in mouse serum. The SNCG expression level of CRC patients treated with bevacizumab combined with chemotherapy was detected by ELISA, and the SNCG level of 5ng / ml was used as the cutoff value. It was found that the progression-free survival (PFS, average 10.5 months) of SNCG-negative patients was significantly longer than that of SNCG-positive patients (average 6.2 months), and the difference between the two was significant (P=0.005). Therefore, anti-SNCG antibodies are expected to be used in the treatment of bevacizumab-resistant tumors, expand the population suitable for anti-angiogenic drug treatment, and synergistically enhance the treatment effect.

[0060] In some embodiments of the present invention, a NOD-SCID mouse subcutaneous tumor model was established using intestinal cancer cells HT29; after tumor formation, bevacizumab or / and SNCG antibody (antibody #1) (100 μg per mouse each time) were administered separately or in combination through the tail vein twice a week; the drug was stopped after 5 administrations, and the mice were euthanized one week after the drug was stopped, and the tumors were removed and weighed. The results showed that the tumor inhibition rates of SNCG antibody or bevacizumab alone were 23.5% and 29.7%, respectively, while the tumor inhibition rate of the combined administration group was 54.9%, which had a significant synergistic tumor inhibition effect.

[0061] In some embodiments of the present invention, a mouse tumor metastasis model is established by injecting intestinal cancer cells HT29 into the tail vein of mice. The next day, tail vein administration (divided into control, SNCG antibody (1# antibody), bevacizumab and SNCG antibody (1# antibody) + bevacizumab combination, a total of 4 groups, 100 μg / time) was started twice a week for a total of 10 times. On the 50th day after administration, the mice were euthanized, and the metastasis of tumor cells in the mice was examined and compared. The results showed that SNCG antibody alone had no obvious inhibitory effect on the in vivo metastasis of HT29 cells. Although bevacizumab had a certain degree of anti-metastatic effect, it did not reach statistical significance; the combined application group of SNCG antibody and bevacizumab had a significant synergistic inhibitory effect on tumor metastasis.

[0062] In some embodiments of the present invention, HT29 cells were injected into the tail vein of mice, and the tail vein administration was started the next day (divided into control, 1#SNCG antibody alone, bevacizumab alone, 1#SNCG antibody + bevacizumab, a total of 4 groups; 100 μg / mouse / time), twice a week, and the survival of mice in each group was observed after 10 administrations. The results showed that there was no significant difference in the survival of mice between the bevacizumab group alone or the SNCG monoclonal antibody group alone and the control group, while the survival of mice in the SNCG antibody and bevacizumab combined administration group was significantly prolonged.

[0063] According to another aspect of the present invention, the present invention also provides a bifunctional antibody against SNCG and VEGF, which is formed by coupling the heavy chain C-terminus of the SNCG antibody of the present invention to a bevacizumab single-chain antibody or a VEGF nanobody. The SNCG antibody can be directly coupled to the bevacizumab single-chain antibody or the VEGF nanobody, or coupled through a connecting peptide segment, etc. In some embodiments of the present invention, at the C-terminus of the heavy chain of the humanized SNCG antibody, the cDNA expressing the bevacizumab single-chain antibody or the cDNA expressing the anti-VEGF nanobody is recombinantly expressed at the gene level to obtain a bifunctional antibody against SNCG and VEGF; ELISA detection shows that the antibody can simultaneously recognize SNCG and VEGF, and can block the binding of VEGF to VEGFR. In some specific embodiments of the present invention, a variety of anti-SNCG / VEGF bifunctional antibodies are provided, which are named 5#, 6#, 7#, and 8# respectively. In some specific embodiments, the anti-SNCG / VEGF bifunctional antibodies 5#, 6#, 7#, and 8# can both bind to SNCG and react with VEGF to varying degrees.

[0064] In some embodiments of the present invention, the anti-migration and invasion activity of the bifunctional antibody was detected by transwell assay, and the results showed that the obtained bifunctional antibody can significantly inhibit the migration and invasion of cells, and its inhibitory effect is significantly higher than that of using SNCG antibody or bevacizumab alone, and is equivalent to the combined use of SNCG antibody and bevacizumab.

[0065] In some embodiments of the present invention, a tumor-bearing mouse model was established using intestinal cancer cells DLD-1-SNCG that overexpress SNCG. After tumor formation, PBS, bevacizumab (Bev) and bifunctional antibody (5#) were respectively administered through the tail vein, twice a week; after 8 administrations, the drug was stopped, the mice were euthanized, and the tumors were removed and weighed. The results showed that the tumor inhibition rate of the bevacizumab group was 27.89%, which was not significantly different from that of the PBS control group. The tumor inhibition rate of the bifunctional antibody group was 52.73%, which was significantly different. The results show that SNCG-high-expressing tumor cells will produce treatment resistance to bevacizumab, and bifunctional antibodies can effectively overcome this problem and improve the therapeutic effect of bevacizumab on drug-resistant tumor cells.

[0066] In some embodiments of the present invention, a NOD-SCID mouse subcutaneous tumor model was established with intestinal cancer cells HT29; after tumor formation, PBS, bevacizumab (Bev), humanized anti-SNCG antibody (1#), bifunctional antibody (5#), and bevacizumab (Bev) + humanized anti-SNCG antibody (1#) were administered through the tail vein, twice a week; after 5 administrations, the drug was stopped, the mice were euthanized, and the tumors were removed and weighed. The results showed that the tumor inhibition rates of SNCG antibody or bevacizumab alone were 15.13% and 36.09%, respectively, while the tumor inhibition rates of the combined administration group were 46.62%, and the tumor inhibition rates of the bifunctional antibody group were 50.34%. The combined administration group and the bifunctional antibody group had a significant synergistic tumor inhibition effect.

[0067] According to another aspect of the present invention, the present invention also provides a nucleic acid molecule encoding the SNCG binding molecule of the present invention (eg, the SNCG antibody or a biologically active fragment derived from the antibody that can specifically bind to SNCG).

[0068] According to another aspect of the present invention, the present invention also provides a vector containing the above-mentioned nucleic acid molecule of the present invention.

[0069] According to another aspect of the present invention, the present invention also provides a cell containing the above nucleic acid molecule of the present invention or containing the above vector.

[0070] According to another aspect of the present invention, the present invention also provides a pharmaceutical composition, which comprises the SNCG binding molecule described in the present invention (such as an SNCG antibody or a biologically active fragment derived from the antibody that can specifically bind to SNCG) or the anti-SNCG and VEGF bifunctional antibody, and a pharmaceutically acceptable carrier or excipient.

[0071] According to another aspect of the present invention, the present invention also provides the use of the SNCG binding molecule or the anti-SNCG and VEGF bifunctional antibody or the pharmaceutical composition in the preparation of a therapeutic agent for treating a disease in a subject, wherein the disease includes a disease related to SNCG expression in cells and tissues.

[0072] According to another aspect of the present invention, the present invention also provides the use of the SNCG binding molecule in the preparation of a diagnostic agent for diagnosing a disease in a subject, wherein the disease includes a disease associated with SNCG expression in cells and tissues.

[0073] According to a specific embodiment of the present invention, the SNCG binding molecule of the present invention can be used alone as an active ingredient or in combination with other active ingredients. For example, the SNCG binding molecule can be used to prepare a bispecific antibody or an antibody-drug conjugate (ADC).

[0074] According to a specific embodiment of the present invention, in the present invention, the disease associated with the expression of SNCG in cells and / or tissues includes cancer. Specifically, the cancer includes but is not limited to: intestinal cancer.

[0075] In summary, the present invention provides a SNCG antibody, which can be used to treat bevacizumab-resistant tumors, expand the population applicable to anti-angiogenic drug treatment, and synergistically enhance the treatment effect. BRIEF DESCRIPTION OF THE DRAWINGS

[0076] Figure 1 The detection results of each SNCG antibody binding reaction are shown.

[0077] Figure 2 The results showed that the ability of 1#SNCG monoclonal antibody (αSNCG) and bevacizumab (Bev) to inhibit the growth of subcutaneous HT29 tumors in mice was significantly higher than that of any monoclonal antibody. In Figure A, the inhibition rate of αSNCG and Bev on HT29 tumor growth was 54.9%, which was higher than that of the αSNCG (23.5%) and Bev (29.7%) groups alone; Figure B is a bar graph of Figure A, and there are statistically significant differences between the combination of αSNCG and Bev and the control, αSNCG alone, and Bev; Figure C shows the volume changes of subcutaneous tumors in mice during tumor formation and drug administration.

[0078] Figure 3 The results show that the ability of 1#SNCG monoclonal antibody (αSNCG) and bevacizumab (Bev) to inhibit the metastasis of HT29 tumor cells in mice is significantly higher than the effect of either monoclonal antibody alone. In Figure A, the inhibitory effect of αSNCG and Bev on HT29 metastasis is much higher than the effect of αSNCG or Bev alone; Figure B is a bar graph of Figure A.

[0079] Figure 4 It shows that in the mouse tumor metastasis model, the combination of 1#SNCG antibody and bevacizumab can significantly prolong the survival of mice. In Figure A, 8×10 5 HT29 cells, 6 mice in each group, were administered via tail vein the next day, twice a week, for 8 consecutive weeks, and the survival time of mice in each group was compared. Image B: Comparison of the 1#SNCG monoclonal antibody (αSNCG) + bevacizumab (Bev) combined administration group and the control group.

[0080] Figure 5 The results show that the humanized anti-VEGF / SNCG bifunctional antibody expressed by genetic engineering can bind to VEGF and SNCG at the same time, and can block the binding of VEGF to VEGFR1 and VEGFR2. Among them, 1μg / ml SNCG (Picture A) was coated on the ELISA enzyme-labeled plate, and the above antibodies were added respectively, reacted at room temperature for 1 hour, and then VEGF was added to detect the binding ability of VEGF. 1μg / ml VEGFR1 (Picture B) and 5μg / ml VEGFR2 (Picture C) were coated on the ELISA enzyme-labeled plate respectively, and the above antibodies and VEGF were added at room temperature for 1 hour to detect the binding level of VEGF to VEGFR1 (Picture B) and VEGFR2 (Picture C) in the presence of each antibody.

[0081] Figure 6 The bifunctional antibody against VEGF / SNCG showed that the inhibitory effect on cell migration and invasion was significantly higher than that of bevacizumab alone, SNCG antibody or the combination of the two. Among them, the cell migration and invasion experiments were conducted in the presence of 20 μg / ml of the above antibodies in the human colon cancer cell line LOVO-SNCG (LOVO cells stably transfected with SNCG) (Picture A, Picture B) and the SNCG-positive cell line SW480 (Picture C, Picture D).

[0082] Figure 7 It was shown that the SNCG / VEGF bifunctional antibody significantly inhibited the tumor growth of DLD1-SNCG tumor-bearing mice with high SNCG expression.

[0083] Figure 8 It was shown that SNCG / VEGF bifunctional antibody inhibited the growth of subcutaneous HT29 tumors in mice. DETAILED DESCRIPTION

[0084] In order to have a clearer understanding of the technical features, purposes and beneficial effects of the present invention, the technical scheme of the present invention is described in detail below in conjunction with specific embodiments and accompanying drawings. Those skilled in the art will appreciate that the embodiments describe the present invention by way of example and are not intended to limit the scope of protection claimed in the present invention. The technical features involved in each embodiment of the present invention can be combined with each other as long as they do not conflict with each other. All public cases and other references mentioned herein are incorporated herein by reference in their entirety. In the embodiments, each raw reagent material is commercially available, and the experimental method without specifying specific conditions is a conventional method and conventional conditions well known in the art, or according to the conditions recommended by the manufacturer. The reagents or instruments used without specifying the manufacturer are all conventional products that can be obtained commercially.

[0085] Example 1: SNCG monoclonal antibody (αSNCG), humanization, preparation and detection

[0086] 1. Mouse SNCG monoclonal antibody

[0087] Construct the GST-SNCG fusion protein, express and purify it in Escherichia coli, and then immunize 6-8 week old BALB / c mice. Take 50μg of GST-SNCG protein and inject it subcutaneously at multiple points on the back with Freund's complete adjuvant (Sigam) to complete the initial immunization. Three weeks later, take 50μg of GST-SNCG protein and inject it subcutaneously at multiple points on the back with Freund's incomplete adjuvant (Sigam) to complete the second immunization. Three weeks later, take 50μg of GST-SNCG protein and inject it subcutaneously at multiple points on the back with Freund's incomplete adjuvant to complete the third immunization. Two weeks later, take 50μg of GST-SNCG protein and inject it intraperitoneally for a booster immunization. Take the mouse spleen 3 days after the booster immunization and perform a fusion experiment with SP 2 / 0 myeloma cells.

[0088] Mouse spleen was taken to prepare spleen cell suspension. Myeloma cells and spleen cells were mixed at a ratio of 1:10 and fused using 50% PEG-DMEM (PEG purchased from Fluka). The fused cells were plated on a 96-well plate and cultured in an incubator at 37°C and 5% CO2. After 7-10 days, clones were grown and tested.

[0089] Use GST / GST-SNCG to coat a 96-well plate, add 50 μL / well of the hybridoma cell supernatant to the prepared 96-well plate, and incubate at 4°C overnight. After washing, add HRP-labeled goat anti-mouse secondary antibody (Jackson ImmunoResearch) and incubate at room temperature for 1 hour. After washing, add OPD for color development. Use 12.5% ​​H 2 SO 4 After the reaction was terminated, the OD490 was read by MicroPlate Reader. The clones that reacted positively with GST-SNCG and negatively with GST were candidate clones.

[0090] In this embodiment, a mouse SNCG monoclonal antibody was screened and obtained. After sequencing and identification, the amino acid sequences of the heavy chain variable regions HCDR1, HCDR2 and HCDR3 of the antibody were: GYTFTDYAIH, AIDPETGGTAYNQKFKG and VAY, respectively; the amino acid sequences of the light chain variable regions LCDR1, LCDR2 and LCDR3 of the antibody were: KSSQSLLDSDGKTYLN, LVSKLDS and WQGTHFPQT, respectively.

[0091] 2. Antibody Humanization

[0092] The variable region sequence of the mouse SNCG antibody obtained by sequencing was inserted into the human IgG1 antibody expression vector to construct the SNCG humanized antibody expression vector and perform antibody humanization. At the same time, mutation sites were introduced at different sites of the IgG1 vector to prepare mutants. The constructed SNCG humanized antibody expression vector was transfected into CHO cells to express the SNCG humanized antibody. After the expressed antibody was purified, its expression efficiency and affinity changes were tested. After design optimization, the optimal humanized antibody was finally selected based on the functional activity, stability, homogeneity of the antibody and the absence of post-translational modifications in the variable region.

[0093] In this example, four humanized SNCG antibodies were obtained, and their variable region amino acid sequences are as follows (CDR sequences are shown in bold and underlined):

[0094] Heavy chain of humanized SNCG antibody 1#:

[0095]

[0096] Light chain of humanized SNCG antibody 1#:

[0097]

[0098] Heavy chain of humanized SNCG antibody 2#:

[0099]

[0100] Light chain of humanized SNCG antibody 2#:

[0101]

[0102] Heavy chain of humanized SNCG antibody 3#:

[0103]

[0104] Light chain of humanized SNCG antibody 3#:

[0105]

[0106] Heavy chain of humanized SNCG antibody 4#:

[0107]

[0108] Light chain of humanized SNCG antibody 4#:

[0109]

[0110]

[0111] 3. Humanized Antibody Detection

[0112] In this example, the binding activity of the obtained four different forms of humanized antibodies (labeled as 1#, 2#, 3#, and 4#) was detected by ELISA method. Bevacizumab (Bev) and irrelevant humanized antibody 101 were used as negative controls, and αSNCG was used as SNCG positive control antibody.

[0113] ELISA test method for the reactivity of each antibody: 1 μg / ml GST-SNCG, GST-SNCA, GST-SNCB were coated on ELISA plates respectively, overnight at room temperature; washed 3 times with PBST; 0.5 μg / ml of each antibody of αSNCG, Bev, 1#, 2#, 3#, 4#, 101 were added, reacted at room temperature for 1 hour; washed 3 times with PBST; rabbit anti-human or anti-mouse enzyme-labeled secondary antibody was added, reacted at room temperature for 1 hour; washed 3 times with PBST; substrate TMB was added for color development for 15 minutes, 2M H 2 SO 4 Terminate the reaction; measure OD value at 450nm.

[0114] Similarly, the purified SNCG protein was coated on an ELISA plate to detect the reactivity of each antibody.

[0115] Results Figure 1 The results showed that αSNCG, humanized antibodies 1#, 2#, 3#, and 4# specifically bound to SNCG, but had no reaction with SNCA and SNCB, and Bev had no reaction with SNCG, SNCA, and SNCB (Picture A); the same result was obtained when SNCG was used alone for coating (Picture B).

[0116] Example 2: The ability of 1#SNCG monoclonal antibody (αSNCG) combined with bevacizumab (Bev) to inhibit the growth of subcutaneous HT29 tumors in mice is significantly higher than that of either monoclonal antibody

[0117] HT29 is a tumor cell line insensitive to bevacizumab. To confirm the effect of SNCG antibody in bevacizumab-insensitive tumor cells, the following experiment was performed.

[0118] Experimental method: HT29 cells were inoculated subcutaneously in the right upper axilla of NOD-SCID mice, 2×10 6 cells / mouse, including control group, SNCG antibody group (1#antibody, αSNCG, 5 mg / kg), bevacizumab (Roche, 100 mg / bottle) treatment group (Bev, 5 mg / kg) and the combination group, a total of 4 groups, 6 mice in each group; when the subcutaneous tumor volume reached 100mm 3 Around 24 hours after the onset of the disease, bevacizumab was administered by tail vein twice a week. The length and width of the tumors were measured and the body weight was weighed. When the maximum tumor volume reached 1000 mm 3At about 12 h, the mice were euthanized, the tumors were removed and weighed, and the tumor inhibition rates were statistically analyzed.

[0119] Results Figure 2 The results showed that the inhibition rate of αSNCG combined with Bev on HT29 tumor growth was 54.9%, which was higher than the inhibition rate of αSNCG (23.5%) and Bev (29.7%).

[0120] Example 3: The ability of 1#SNCG monoclonal antibody (αSNCG) and bevacizumab (Bev) to inhibit the metastasis of HT29 tumor cells in mice (A, B) is significantly higher than the effect of either monoclonal antibody alone

[0121] This example further studies the effect of the combined use of SNCG antibody and bevacizumab on tumor metastasis.

[0122] Experimental method: Each NOD-SCID mouse was injected with 8×10 5 HT29 cells, 6 mice in each group, including control group, 1#SNCG antibody group (αSNCG, 5 mg / kg), bevacizumab treatment group (Bev, 5 mg / kg) and the combination group, 100 μg / mouse was administered through the tail vein the next day, twice a week, for 5 consecutive weeks, during which the mice were weighed, and after obvious metastases appeared under the skin of the mice, the mice were euthanized, the metastases were removed and weighed, and the number of metastases in each group of mice was compared.

[0123] Results Figure 3 The results showed that the ability of the combination of 1#SNCG monoclonal antibody (αSNCG) and bevacizumab (Bev) to inhibit the metastasis of HT29 tumor cells in mice was significantly higher than that of either monoclonal antibody alone, and there was a statistically significant difference between the combination of αSNCG and Bev and the control, and between αSNCG and Bev alone.

[0124] Example 4: In a mouse tumor metastasis model, the combination of 1#SNCG antibody (αSNCG) and bevacizumab antibody can significantly prolong the survival of mice

[0125] This example further verifies the effects of 1#SNCG monoclonal antibody (αSNCG) and bevacizumab (Bev) on the survival of mice in tumor metastasis.

[0126] Experimental method: Intestinal cancer cell HT29 is a cell with high endogenous SNCG expression and is inherently resistant to bevacizumab. In this experiment, each NOD-SCID mouse was injected with 8×10 5HT29 cells were used in the experiment. The experimental groups included a control group, a 1#SNCG antibody group (αSNCG, 5 mg / kg), a bevacizumab treatment group (Bev, 5 mg / kg), and a combination group of the two. There were 6 mice in each group, for a total of 4 groups. The drugs were administered through the tail vein the next day, twice a week, for 8 consecutive weeks. The survival status and weight of the mice were recorded, and the survival time of the mice in each group was compared.

[0127] Results Figure 4 As shown. The results show that compared with the control group, single antibody 1#SNCG monoclonal antibody (αSNCG) or bevacizumab (Bev) has no significant effect on prolonging the generation period of mice, but the combination of the two can significantly prolong the survival time of mice with metastatic tumors (Picture A). Picture B is a comparison between the αSNCG and Bev combined group and the negative control group. It can be seen that the combined administration can significantly prolong the survival time of mice with endogenous SNCG positive cell HT29 metastasis. The horizontal axis in the figure represents the number of days the tumor metastasis mice survive.

[0128] Example 5: Humanized anti-VEGF / SNCG bifunctional antibody expressed by genetic engineering can bind to VEGF and SNCG simultaneously and block the binding of VEGF to VEGFR1 and VEGFR2

[0129] The combination of αSNCG and Bev based on the present invention can significantly inhibit the metastasis of HT29 tumor cells in mice, and significantly prolong the survival time of mice bearing endogenous SNCG-positive colorectal cancer cells HT29 and mice bearing LOVO-SNCG-transferred colorectal cancer cells. In this example, a SNCG / VEGF bifunctional antibody was prepared, and its biological activity and effect on the phenotype of tumor cells were identified.

[0130] Preparation of SNCG / VEGF bifunctional antibody: The C-terminus of the heavy chain of the humanized SNCG antibody is recombined with the cDNA expressing bevacizumab single-chain antibody or the cDNA expressing anti-VEGF nanoantibody at the gene level, and the bifunctional antibody is expressed after transfection into CHO cells, and the bifunctional antibody of SNCG and VEGF is obtained by purification.

[0131] In this example, four bifunctional antibodies of SNCG and VEGF were obtained, which were labeled as 5#, 6#, 7#, and 8#, respectively. The four bispecific antibodies were 1#, 2#, 3#, and 4# antibodies fused with the same anti-VEGF nanobody at the C-terminus of the heavy chain. The amino acid sequence of 5# antibody is as follows, and 6#-8# antibodies are not described in detail here.

[0132] 5# Antibody heavy chain amino acid sequence:

[0133] MHSSALLCCLVLLTGVRAQVQLVQSGAEVKKPGASVKVSCKASGYTFTDYAIHWVRQAPGQGLEWIGAIDPETGGTAYNQKFKGRATLTADKSISTAYMELSRLRSDDTAVYYCTIVAYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGGSGGGGSGGGGSGGSTVAAPSGSQVKLVESGGGLVQPGGSLRLSCAASGSISYVPDMHWYRQAPGQQRQLVATITRGGNTMYADSVKGRFTISRDNSKNTLYLQMTSLRAEDTAVYYCNADVWSSVLFKLVEYWGQGTLVTVSS(SEQ ID NO:12)

[0134] 5# Antibody light chain amino acid sequence:

[0135] MHSSALLCCLVLLTGVRADIVITQTPLSLSVTPGQPASISCKSSQSLLDSDGKTYLNWLLQKPGQSPQRLIYLVSKLDSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCWQGTHFPQTFGQGTRLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC(SEQ ID NO:13)

[0136] This example further verifies that the obtained SNCG / VEGF bifunctional antibody can bind to SNCG and VEGF simultaneously, and can inhibit the binding of VEGF to VEGFR1 and VEGFR2, thereby blocking the VEGF-VEGFR signaling pathway.

[0137] Experimental methods:

[0138] 1. Can the SNCG / VEGF bifunctional antibody bind to SNCG and VEGF at the same time?

[0139] 1 μg / ml SNCG was coated on the ELISA plate and incubated overnight at room temperature; washed 3 times with PBST; 0.5 μg / ml of each antibody of αSNCG, Bev, 5#, 6#, 7#, 8#, and 101 was added and reacted at room temperature for 1 hour; washed 3 times with PBST; bio-VEGF (1:5000) was added and reacted at room temperature for 1 hour; washed 3 times with PBST; SA-HRP (1:5000) was added and reacted at room temperature for 25 minutes; TMB substrate was added for color development for 15 minutes, 2M H 2 SO 4 Terminate the reaction; measure OD value at 450nm.

[0140] Results Figure 5 The results showed that: SNCG+αSNCG+VEGF and SNCG+Bev+VEGF had no binding reaction, while SNCG+bifunctional antibodies 5#, 6#, 7#, 8#+VEGF had binding reactions to varying degrees. The irrelevant bifunctional antibody 101 was used as a control and had no reaction with SNCG or VEGF (Figure A).

[0141] II. Experiment on the inhibition of VEGF binding to VEGFR1 and VEGFR2 by SNCG / VEGF bifunctional antibody

[0142] 1μg / ml VEGFR1 and 5μg / ml VEGFR2 were coated on ELISA plates respectively; washed 3 times with PBST; bio-VEGF (1:5000) and 10μg / ml of αSNCG, Bev, 5#, 6#, 7#, 8#, 101 antibodies were added at the same time, and an equal volume of dilution + bio-VEGF (1:5000) was used as a positive control, and reacted at room temperature for 1h; washed 3 times with PBST; SA-HRP (1:5000) was added and reacted at room temperature for 25min; washed 3 times with PBST; substrate TMB was added for color development for 15min, 2M H 2 SO 4 Terminate the reaction; measure OD value at 450nm.

[0143] Results Figure 5The results showed that bevacizumab (Bev), bifunctional antibodies 5#, 6#, 7#, and 8# all had the ability to inhibit the binding of VEGF to VEGFR1 (Picture B) and VEGFR2 (Picture C) to varying degrees; the single SNCG antibody αSNCG and the unrelated bifunctional antibody 101 could not inhibit the binding of VEGF to VEGFR1 (Picture B) and VEGFR2 (Picture C).

[0144] Example 6: The inhibitory effect of SNCG / VEGF bifunctional antibody on cell migration and invasion is significantly higher than that of bevacizumab, αSNCG antibody or the combination of the two alone

[0145] Based on the fact that SNCG / VEGF bifunctional antibodies 5#, 6#, 7#, and 8# can all specifically bind to SNCG, and can simultaneously bind to SNCG and VEGF, and can inhibit the binding of VEGF to VEGFR1 and VEGFR2 to varying degrees, this example further explores the effect of bifunctional antibodies on tumor cell phenotypes.

[0146] Experimental methods:

[0147] 1. Resuscitation of human colon cancer cell line LOVO-SNCG (LOVO stable cell line introduced with SNCG) and SNCG positive cell line SW480;

[0148] 2. When the cell growth density reaches 80-90%, perform cell migration and invasion experiments;

[0149] 3. Add 800 μL of RPMI-1640 culture medium containing 10% FBS to the lower chamber of the Transwell. 5 -1×10 6 Cells / 200 μL serum-free RPMI-1640 culture medium (containing 20 μg / ml SNCG / VEGF bifunctional antibodies 5#, 6#, 7#, 8#, bevacizumab, αSNCG antibody or a combination of the two) were added to the upper chamber and incubated for 24 h for cell migration experiments;

[0150] 4. Add 800 μL of RPMI-1640 culture medium containing 10% FBS to the lower chamber of the Transwell. 5 -1×10 6 Cells / 500 μL serum-free RPMI-1640 culture medium (containing 20 μg / ml SNCG / VEGF bifunctional antibodies 5#, 6#, 7#, 8#, bevacizumab, αSNCG antibody or a combination of the two) were added to the upper chamber covered with matrigelde and incubated for 40 hours for cell invasion assay;

[0151] 5. Fix with ice methanol for 10 min, stain with 0.1% crystal violet for 4 h, wipe off the non-migrated cells on the upper layer of the chamber with a cotton swab, seal the slides with neutral gum, take pictures and count the number of migrated cells.

[0152] Results Figure 6 As shown. The results showed that: SNCG antibody (αSNCG), bevacizumab (Bev), bifunctional antibodies 5#, 6#, 7#, 8# all had different degrees of ability to inhibit the migration (Picture A, Picture C) and invasion (Picture B, Picture D) of tumor cells LOVO-SNCG and SW480. The ability of SNCG antibody αSNCG combined with bevacizumab (Bev) to inhibit tumor cell invasion was stronger than that of any single antibody, and the ability of SNCG / VEGF bifunctional antibody, especially 5#, to inhibit tumor cell invasion was significantly higher than that of αSNCG, bevacizumab (Bev) or the combination of the two.

[0153] Example 7: SNCG / VEGF bifunctional antibody inhibits tumor growth in SNCG-highly expressing tumor cells DLD1-SNCG-bearing mice

[0154] DLD1-SNCG is a colorectal cancer cell that exogenously expresses SNCG. This example uses this cell to construct a tumor-bearing mouse model to evaluate the tumor-suppressing effect of the SNCG / VEGF bifunctional antibody on SNCG-high-expressing tumor cells in vivo.

[0155] Experimental method: DLD1-SNCG cell line was inoculated subcutaneously in the right upper axilla of NOD-SCID mice, 3×10 6 cells / mouse, including control group, bevacizumab treatment group (Bev, 5 mg / kg) and bifunctional antibody group (5#, 5 mg / kg), a total of 3 groups, 6 mice in each group; when the subcutaneous tumor volume reached 100mm 3 Around 1000 mm, the drug was administered via the tail vein twice a week. The length and width of the tumor were measured and the body weight was weighed. 3 At about 10 minutes, the experiment was ended, the tumors were removed and weighed, and the tumor inhibition rate and weight changes of mice were statistically analyzed.

[0156] Results Figure 7 The results showed that the tumor inhibition rate of the Bevacizumab (Bev) experimental group was 27.89%, with no statistical difference. The tumor inhibition rate of the SNCG / VEGF bifunctional antibody group (5#) was 52.73%, with statistical difference.

[0157] Example 8: SNCG / VEGF bifunctional antibody inhibits tumor growth in HT29 tumor-bearing mice

[0158] HT29 is an intestinal cancer cell with high expression of SNCG and insensitive to bevacizumab. On the basis of clarifying that the SNCG / VEGF bifunctional antibody can inhibit the in vivo growth of tumors with high expression of SNCG and resistant to bevacizumab, in this example, the animal model was further used to evaluate the in vivo antitumor effect of the SNCG / VEGF bifunctional antibody on tumor cells (HT29) insensitive to bevacizumab.

[0159] Experimental method: HT29 cells were inoculated subcutaneously into the right upper axilla of NOD-SCID mice, 3×10 6 cells / mouse, including a control group, a humanized SNCG antibody group (1#, 5 mg / kg), a bevacizumab treatment group (Bev, 5 mg / kg), a bifunctional antibody group (5#), and a humanized SNCG antibody group (1#, 5 mg / kg) + bevacizumab treatment group (Bev, 5 mg / kg), a total of 5 groups, with 6 mice in each group; when the subcutaneous tumor volume reached about 100 mm 3 or so, drugs were administered via the tail vein, twice a week, measuring the length and width of the tumor and weighing the mice. When the maximum tumor volume reached about 1000 mm 3 or so, the experiment ended, the tumors were taken and weighed. Statistical analysis was performed on the respective tumor inhibition rates and changes in mouse body weight.

[0160] The results are shown in Figure 8 as follows. Figure 8 . The SNCG / VEGF bifunctional antibody inhibits the growth of HT29 tumors subcutaneously in mice. The results show that the tumor inhibition rate of the humanized SNCG antibody (1#) is 15.13%, which is statistically different from that of the control group. In the bevacizumab (Bev) experimental group, the SNCG / VEGF bifunctional antibody group (5#), and the humanized SNCG antibody (1#) + bevacizumab (Bev) experimental group, there is a more significant effect of inhibiting tumor growth, and the tumor inhibition rates are 36.09%, 50.34%, and 46.62% respectively.

[0161] The descriptions presented in the above exemplary embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to be exhaustive, nor to limit the present invention to the precise forms described. Obviously, many changes and variations are possible for those of ordinary skill in the art according to the above teachings. The selection of the exemplary embodiments and the description are for explaining the specific principles of the present invention and its practical applications, so that other technicians in the art can easily understand, implement, and utilize the various exemplary embodiments of the present invention and their various alternative forms and modified forms. The protection scope of the present invention is intended to be defined by the scope of the claims and their equivalent forms.

Claims

1. A molecule comprising an SNCG antibody or a biologically active fragment derived from the antibody that can immunospecifically bind to SNCG, wherein the amino acid sequence of the six CDRs of the SNCG antibody is include: Heavy chain CDR1: GYTFTDYAIH (SEQ ID NO: 1); Heavy chain CDR2: AIDXETGGTAYNQKFKG (SEQ ID NO: 2), wherein X is P or A; Heavy chain CDR3: VAY; Light chain CDR1: KSSQSLLDSDXKTYLN (SEQ ID NO: 3), wherein X is G or A; Light chain CDR2: LVSKLDS (SEQ ID NO: 4); Light chain CDR3: WQGTHFPQT (SEQ ID NO: 5).

2. The molecule according to claim 1, wherein the biologically active fragment derived from the antibody capable of specifically binding to SNCG comprises the amino acid sequence of the six CDRs of the SNCG antibody.

3. The molecule according to claim 1, wherein the antibody is an animal-derived antibody, a chimeric antibody, a humanized antibody or a human antibody.

4. The molecule according to claim 1, include: (1) a heavy chain variable region, wherein the heavy chain variable region has an amino acid sequence of any one of SEQ ID NO:6, SEQ ID NO:8, and SEQ ID NO:10, or an amino acid sequence with equivalent functions formed by replacing, deleting, or adding one or more amino acids on the basis of these amino acid sequences; and / or (2) a light chain variable region, wherein the light chain variable region has an amino acid sequence of any one of SEQ ID NO:7, SEQ ID NO:9, and SEQ ID NO:11, or an amino acid sequence with equivalent functions formed by replacing, deleting, or adding one or more amino acids on the basis of these amino acid sequences; Preferably, the molecule comprises: The heavy chain variable region has the amino acid sequence shown in SEQ ID NO:6, and the light chain variable region has the amino acid sequence shown in SEQ ID NO:7; or The heavy chain variable region has the amino acid sequence shown in SEQ ID NO:8, and the light chain variable region has the amino acid sequence shown in SEQ ID NO:9; or The heavy chain variable region has the amino acid sequence shown in SEQ ID NO:8, and the light chain variable region has the amino acid sequence shown in SEQ ID NO:11; or The heavy chain variable region has the amino acid sequence shown in SEQ ID NO:10, and the light chain variable region has the amino acid sequence shown in SEQ ID NO:

11.

5. A bifunctional antibody against SNCG and VEGF, which is formed by coupling the heavy chain C-terminus of the molecule according to any one of claims 1 to 4 to a bevacizumab single-chain antibody or a VEGF nanobody.

6. A nucleic acid molecule encoding the amino acid sequence of the molecule according to any one of claims 1 to 4 or the bifunctional antibody according to claim 5.

7. A vector or cell containing the nucleic acid molecule according to claim 6.

8. A pharmaceutical composition comprising the molecule according to any one of claims 1 to 4 or the bifunctional antibody according to claim 5, and a pharmaceutically acceptable carrier or excipient.

9. Use of the molecule according to any one of claims 1 to 4, the bifunctional antibody according to claim 5, or the pharmaceutical composition according to claim 8 in the preparation of a therapeutic agent for treating a disease in a subject, or a diagnostic agent for diagnosing a disease in a subject, in, The diseases include diseases associated with SNCG expression in cells and / or tissues.

10. The use according to claim 9, in, The molecule according to any one of claims 1 to 4 is used alone as an active ingredient or in combination with other active ingredients, wherein the other active ingredient is preferably bevacizumab; Preferably, the molecule according to any one of claims 1 to 4 can be used to prepare a bispecific antibody or an antibody-drug conjugate; Preferably, the disease associated with SNCG expression in cells and / or tissues includes cancer; more preferably, the cancer includes but is not limited to colon cancer.

Images